This invention pertains to the field of genetic engineering. In particular, it relates to the resolution of complex tandem repeats of exogenous polynucleotide sequences into a single copy of a desired exogenous polynucleotide.
Genetic transformation of eukaryotes often results in multiple copies of an exogenous polynucleotide becoming integrated at the same locus. For instance, with current plant transformation methods, the foreign DNA often integrates imprecisely and as multiple copies at a single locus. Often, this results in a long array of transgene copies, not all of which may be full-length or faithful replicas of the introduced DNA (see, e.g. Srivastava, V. et al. Theoret. Appl. Genet. 92: 1031-1037 (1996); Kononov, M. E. et al. Plant J. 11: 945-957 (1997); Iglesias, V. A. et al. Plant Cell 9: 1251-1264 (1997); Kohli, A. et al. Proc. Natl. Acad. Sci. USA 95: 7203-7208 (1998)). A tandem array of like-sequences can lead to DNA rearrangements. Homologous recombination can excise or invert endogenous chromosomal DNA found within the array of introduced polynucleotides. The unequal crossing-over between homologous chromosomes or sister chromatids can alter gene copy number.
These structural changes can affect transgene expression, and may be a significant cause of the transgene instability phenomenon; particularly those manifested in subsequent generations (see, e.g., Cluster, P. D. et al. Plant Mol. Biol. 32: 1197-1203 (1996)). Aside from structural rearrangements, sufficient correlation has indicated that multiple copies of the transgene, by itself, can induce "homology-dependent gene silencing", where gene expression is repressed due to unknown interactions between homologous sequences (see, Kooter, J. M. and Mol, J. N. M., Curr. Opinion Biotechnol. 4: 166-171 (1993); Jorgensen, R. A., Science 268: 686-691 (1995); Meyer, P., and Saedler, H., Annu. Rev. Plant Mol. Biol. 47: 23-48 (1996); Cluster, P. D. et al., Plant Mol. Biol. 32: 1197-1203 (1996)).
For commercial production of a variety of transgenic organisms, the search among transformants for those that have single copy insertions is not only necessary in many cases, but requires a significant effort. For example, in the commercial production of transgenic plants, the prevalence of single copy insertions varies depending on the transformation method and the plant species. With direct DNA transformation methods, such as those reported for the biolistic transformation of wheat, single copy insertions are rarely recovered (see, e.g., Vasil, V. et al., Bio/Technol. 11: 1553-1558 (1993); Weeks, T. J. et al., Plant Physiol. 102: 1077-1084 (1993); Becker, D. et al., Plant J. 5: 299-307(1994); Nehra, N. S. et al., Plant J. 5: 285-297 (1994); Pawlowski, W. and Somers, D., Mol. Biotechnol. 6:17-30 (1996)).
Thus, one problem in genetic engineering is the lack of methods to produce transgenic organisms that have only a single copy of a transgene. The invention presented herein addresses this and other problems.